Specific antiviral therapy for influenza is urgently needed to complement vaccination and strengthen global efforts to control potential new pandemic. Development of new anti-influenza treatments is critical also due to emerging antiviral resistance to existing drugs. The long-term objective of this research plan is to develop a safe and highly effective intranasal hemagglutinin (HA) derived inhibitor as prophylaxis for use in high-risk unvaccinated populations. In preliminary work, we have generated influenza fusion inhibitors by conjugating peptides derived from the C-terminal region of HA and by adding a cell penetrating peptide sequence. We have shown that prophylactic intranasal administration of our lead HA fusion inhibitor is as efficient as treatment with approved drug Relenza(c) at inhibiting viral replication in vivo. The HA fusion inhibitor peptides self-assemble in stable ~100nm nanoparticles until they reach the target cells where they are integrated into cell membranes. These peptides are easy to prepare and have a well defined scheme allowing several design alternatives. We propose to optimize: 1) the antiviral potency, 2) the conditions under which the peptides self-assemble, 3) the mechanical properties critical for handling and intracellular delivery, 4) the insertion on the target cell membrane, and 5) in vivo biodistribution. This unique combination of properties will be developed in this application to delivery and to create a protective shield on the human airway epithelium to prevent viral infection. To achieve our goals we propose two aims: 1. To use structure- guided mutagenesis and protein engineering to identify and develop optimized HA peptide fusion inhibitors. A systematic structural approach will identify and incorporate specific residue substitutions at the inhibitor- binding interface to impart additional binding energy of the peptide to its target region on HA. Specific mutations to increase protease resistance will be evaluated. Biophysical analysis will identify linker length and lipid moiety that provide optimal balance between self-assembling prowess, nanoparticles stability, and homogeneity. The improved nanoparticles will be tested in vitro and ex vivo to assess their antiviral activity. 2. To evaluate the protection afforded by optimized self-assembling nanoparticles against HA infection in cotton rat. We will evaluate the biodistribution and toxicity properties of the optimized nanoparticles, and use cotton rat model to assess their in vivo anti-influenza potency. In an iterative process, the outcome of the experiments will guide further optimization, yielding a set of promising investigational anti-influenza agents.